This invention is directed to test strips or assay devices for determination of characteristics of samples, unitized housings, and kits incorporating test strips and housings, and methods of determining the characteristics of samples using the test strips and housings, particularly for the performance of competitive assays.
Among the many analytical systems used for detection and/or determination of analytes, particularly analytes of biological interest, are chromatographic assay systems. Among the analytes frequently assayed with such systems are:
(1) hormones, such as human chorionic gonadotropin (hCG), frequently assayed as a marker of human pregnancy; PA1 (2) antigens, particularly antigens specific to bacterial, viral, and protozoan pathogens, such as Streptococcus, hepatitis virus, and Giardia; PA1 (3) antibodies, particularly antibodies induced as a result of infections with pathogens, such as antibodies to the bacterium Helicobacter pylori and to human immunodeficiency virus (HIV); PA1 (4) other proteins, such as hemoglobin, frequently assayed in determinations of fecal occult blood, an early indicator of gastrointestinal disorders such as cancer; PA1 (5) enzymes, such as aspartate aminotransferase, lactate dehydrogenase, alkaline phosphatase, and glutamate dehydrogenase, frequently assayed as indicators of physiological function and tissue damage; PA1 (6) drugs, both therapeutic drugs, such as antibiotics, tranquilizers, and anticonvulsants, and illegal drugs of abuse, such as cocaine, heroin, and marijuana; PA1 (7) environmental pollutants such as pesticides and aromatic hydrocarbons; and PA1 (8) vitamins. PA1 (1) a first opposable component including: PA1 (2) a second opposable component hingedly attachable to the first opposable component including: PA1 (1) the immunoassay device described above; and PA1 (2) a liquid for resolubilizing the labeled specific binding partner to the analyte or analog thereof in the comparison label zone. PA1 (1) adding a test sample to the sample preparation zone of the competitive immunoassay device described above; PA1 (2) allowing the test sample to resolubilize the labeled specific binding partner for the analyte conjugated to the first member of the auxiliary specific binding pair; PA1 (3) adding a liquid to the comparison label zone to resolubilize the labeled specific binding partner to the analyte or analog thereof; PA1 (4) bringing the first and second opposable components into opposition from a position in which they are not in opposition to bring the absorber on the first opposable component into operable contact with the second end of the first chromatographic medium and the second end of the second chromatographic medium and to bring the sample preparation zone into operable contact with the first end of the first chromatographic medium; PA1 (5) allowing the sample and the labeled specific binding partner for the analyte conjugated to the first member of the auxiliary specific binding pair to flow through the first chromatographic medium and the labeled specific binding partner to the analyte or analog thereof to flow through the second chromatographic medium so that, if analyte is present in the test sample, the labeled specific binding partner to the analyte conjugated to the first member of the auxiliary specific binding pair binds to the detection zone on the first chromatographic medium and so that the labeled specific binding partner to the analyte or analog thereof binds to the comparison zone on the second chromatographic medium; and PA1 (6) comparing the intensity of label at the detection zone on the first chromatographic medium and at the comparison zone on the second chromatographic medium to detect and/or determine the analyte in the test sample. PA1 (1) a first opposable component including: PA1 (2) a second opposable component including a chromatographic medium with first and second ends, the chromatographic medium including thereon in discrete, nonoverlapping zones: PA1 (1) applying the sample to the sample preparation zone of the single-chromatographic medium competitive immunoassay device described above; PA1 (2) allowing the sample applied to the sample preparation zone to resolubilize the labeled specific binding partner for the analyte conjugated to the first member of the auxiliary specific binding pair and the predetermined quantity of the analyte or analog thereof covalently bound to the labeled specific binding partner for the molecule that does not substantially cross-react with the analyte; PA1 (3) bringing the first and second opposable components into opposition from a position in which they are not in opposition so that the absorber is brought into operable contact with the second end of the chromatographic medium and so that the sample preparation zone is brought into operable contact with the first end of the chromatographic medium; PA1 (4) allowing the sample, the labeled specific binding partner for the sample, and the analyte or analog thereof covalently bound to the labeled specific binding partner for the molecule that does not substantially cross-react with the analyte to migrate through at least a portion of the chromatographic medium including the detection zone and the control zone; and PA1 (5) detecting and/or determining the concentration of analyte in the test sample by comparing the intensities of label at the detection zone and the control zone of the chromatographic medium. PA1 (1) a first opposable component including: PA1 (2) a second opposable component hingedly attachable to the first opposable component including: PA1 (1) the immunoassay device; and PA1 (2) a liquid for resolubilizing the labeled specific binding partner to the analyte or analog thereof in each comparison label zone. PA1 (1) adding a test sample to at least one of the sample preparation zones of the multiplex assay device; PA1 (2) allowing the at least one test sample to resolubilize the labeled specific binding partners for the analyte conjugated to the first member of the auxiliary specific binding partner; PA1 (3) adding a liquid to at least one of the comparison label zones to resolubilize the labeled specific binding partner to the analyte or analog thereof; PA1 (4) bringing the first and second opposable components into opposition from a position in which they are not in opposition to bring the absorber on the first opposable component into operable contact with the second end of the first chromatographic media and the second end of the second chromatographic media and to bring the sample preparation zones into operable contact with the first ends of the first chromatographic media; PA1 (5) allowing the at least one sample and the labeled specific binding partner for the analyte conjugated to the first member of the auxiliary specific binding pair to flow through the first chromatographic media and the labeled specific binding partner to the analyte or analog thereof to flow through the second chromatographic media so that, if analyte is present in any test sample, the labeled specific binding partner to the analyte conjugated to biotin binds to the detection zone on the corresponding first chromatographic medium and so that the labeled specific binding partner binds to the comparison zone on the corresponding second chromatographic medium; and PA1 (6) comparing the intensity of label at the detection zone on each first chromatographic medium and at the comparison zone on each second chromatographic medium to detect and/or determine the analyte in each test sample. PA1 (1) a first opposable component including: PA1 (2) a second opposable component including a plurality of chromatographic media with first and second ends, each chromatographic medium including thereon in discrete, nonoverlapping zones: PA1 (1) applying a sample to at least one of the sample preparation zones of the multiplex assay device; PA1 (2) allowing the at least one sample applied to the sample preparation zones to resolubilize the labeled specific binding partner for the analyte conjugated to the first member of the auxiliary specific binding pair and the predetermined quantity of the analyte or analog thereof covalently bound to the labeled specific binding partner for the molecule that does not substantially cross-react with the analyte; PA1 (3) bringing the first and second opposable components into opposition from a position in which they are not in opposition so that the absorber is brought into operable contact with the second end of each chromatographic medium and so that each sample preparation zone is brought into operable contact with the first end of each chromatographic medium; PA1 (4) allowing the at least one sample, the labeled specific binding partner for the sample, and the analyte or analog thereof covalently bound to the labeled specific binding partner for the molecule that does not substantially cross-react with the analyte to migrate through at least a portion of each chromatographic medium including the detection zone and the control zone; and PA1 (5) detecting and/or determining the concentration of analyte in each test sample by comparing the intensities of label at the detection zone and the control zone of each chromatographic medium.
Such chromatographic systems are frequently used by physicians and medical technicians for rapid in-office diagnosis and therapeutic monitoring of a variety of conditions and disorders. They are also increasingly used by patients themselves for at-home monitoring of such conditions and disorders.
Among the most important of such systems are the "thin layer" systems in which a solvent moves across a thin, flat absorbent medium.
Among the most important of tests that can be performed with such thin layer systems are immunoassays, which depend on the specific interaction between an antigen or hapten and a corresponding antibody. The use of immunoassays as a means of testing for the presence and/or mount of clinically important molecules has been known for some time. As early as 1956, J. M. Singer reported the use of an immune-based latex agglutination test for detecting a factor associated with rheumatoid arthritis (J. M. Singer et al., Am. J. Med. 22:888-892 (1956)).
Among the chromatographic techniques used in conjunction with immunoassays is a procedure known as immunochromatography. In general, this technique uses a disclosing reagent or particle that has been linked to an antibody to the molecule to be assayed, forming a conjugate. This conjugate is then mixed with a specimen and, if the molecule to be assayed is present in the specimen, the disclosing reagent-linked antibodies bind to the molecule to be assayed, thereby giving an indication that the molecule to be assayed is present. The disclosing reagent or particle can be identifiable by color, magnetic properties, radioactivity, specific reactivity with another molecule, or another physical or chemical property. The specific reactions that are employed vary with the nature of the molecule being assayed and the sample to be tested.
Immunochromatographic assays fall into two principal categories: "sandwich" and "competitive," according to the nature of the antigen-antibody complex to be detected and the sequence of reactions required to produce that complex. The antigen to be detected can itself be an antibody, such as serological assays for H. pylori-specific antibody. In such cases, the antibody to be detected can be bound to a specific antigen. Alternatively, the antigen to be detected can be detected indirectly by using a labeled second antibody that binds to the first antibody to the analyte to be detected.
In competitive immunoassays, the label is typically a labeled analyte or analyte analog which competes for binding of an antibody with any unlabeled analyte present in the sample. Competitive immunoassays are typically used for detection of analytes such as haptens, each hapten being monovalent and capable of binding only one antibody molecule. Examples of competitive immunoassay devices are those disclosed by U.S. Pat. No. 4,235,601 to Deutsch et al., U.S. Pat. No. 4,442,204 to Liotta, and U.S. Pat. No. 5,208,535 to Buechler et al., all which are incorporated herein by this reference.
Although useful, currently available chromatographic techniques using test strips have a number of drawbacks. Many samples, such as fecal samples, contain particulate material that can clog the pores of the chromatographic medium, greatly hindering the immunochromatographic process. Other samples, such as blood, contain cells and colored components that make it difficult to read the test. Still other samples, such as milk, contain fat globules or other components that can create interference. Even if the sample does not create interference, it is frequently difficult with existing chromatographic test devices to apply the sample to the chromatographic medium so that the sample front moves uniformly through the chromatographic medium to insure that the sample reaches the area where binding is to occur in a uniform, straight-line manner.
Other problems exist with currently-available test strips because of the nature of the sample to be assayed or the assay to be carried out. With such devices, it is impractical to perform washing steps which are frequently desirable to improve sensitivity and to reduce to background. Also, it is difficult, and in many cases impossible, to carry out preincubation steps within the device. Additionally, there is a need for an immunochromatographic assay device that can carry out a broad range of separations, such as a separation of fat from milk or the separation of organic chemicals such as the separation of benzene from toluene.
Sample preparation and waste generation are responsible for other problems with currently available devices and techniques for immunochromatography. The increased prevalence of diseases spread by infected blood and blood fractions, as well as other bodily secretions, such as AIDS and hepatitis, has exacerbated these problems. It is rarely possible to apply a sample (such as feces) or a sampling device (such as a throat swab) directly to the chromatographic medium. Several extraction and pretreatment reactions are usually required before the sample can be applied to the chromatographic medium. These reactions are typically carried out by the physician or technician performing the test in several small vessels, such as test tubes, or microfuge tubes, requiring the use of transfer devices, such as pipettes. Each of these devices is then contaminated and must be disposed of using special precautions so that workers or people who may inadvertently come into contact with the waste do not become contaminated.
Additionally, currently available test devices, although useful, are generally not suited to give a semi-quantitative or quantitative indication of the concentration of an analyte present in a test sample, particularly for competitive immunoassays. Typically, the obtaining of such a quantitative or semi-quantitative indication requires the use of more than one test device, such as for calibration. The use of more than one test device requires a greater expenditure of time and material and also increases the possibility that a mistake may be made during the performance of the assay. In particular, with possibly contaminated samples, there may be concern that, inadvertently, a device thought to contain only a control actually contains a sample that may contain an infectious agent. Thus, it would be preferable to be able to determine a quantitative or semi-quantitative indication of analyte concentration in one assay device without the use of a separate assay device as a control or for calibration.
There is further need for an assay device that gives a positive indication that the test has been performed correctly and that flow has occurred properly within the chromatographic assay device.
Accordingly, there is a need for an improved assay device capable of handling a broad range of chromatographic assays. Such a device should be able to handle all types of immunoassays, particularly competitive immunoassays, as well as other types of assays using chromatography. Such a device should be capable of receiving a possibly contaminated sample or a sample preparation device directly so as to eliminate the need for extraction vessels and transfer devices. Such a device, particularly in the form of a test strip, should also be capable of performing immunochromatographic assays on colored samples or samples containing particulates without interference and should be able to deliver the sample to the chromatographic medium uniformly and evenly to improve accuracy and precision of the tests. Additionally, such an improved test strip should be capable of carrying out semi-quantitative or quantitative indications of analyte concentration in a single assay device without the need for additional control assay devices, and should give a positive indication that flow within the device has occurred properly and that the assay has operated correctly.